LOCUS Exported 6454 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION mammalian expression of Zdk1 tagged RhoA Q63L. ACCESSION . VERSION . KEYWORDS pTriEx-mCherry-Zdk1-RhoA Q63L SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6454) AUTHORS Wang H, Vilela M, Winkler A, Tarnawski M, Schlichting I, Yumerefendi H, Kuhlman B, Liu R, Danuser G, Hahn KM TITLE LOVTRAP: an optogenetic system for photoinduced protein dissociation. JOURNAL Nat Methods. 2016 Jul 18. doi: 10.1038/nmeth.3926. PUBMED 27427858 REFERENCE 2 (bases 1 to 6454) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6454 /organism="synthetic DNA construct" /mol_type="other DNA" misc_recomb 110..937 /label=baculovirus recombination region (lef2/ORF603) /note="contains ORF603 and part of lef2" CDS 110..234 /codon_start=3 /product="baculovirus late expression factor 2" /label=lef2 /translation="HLLIKSQDVYKPPNCQKMKTVDKLCPFAGNCKGLNPICNY" CDS complement(272..877) /codon_start=1 /product="baculovirus ORF603 protein" /label=ORF603 /translation="MAVIFNNKQLLADNSIEKGGELFLFNGSYNILESYVNPVLLKNGV IELEEAAYYAGNILYKTDDPKFIDYINLIIKATHSEELPENSTVVNYRKTMRSGTIHPI KKDIYIYDNKKFTLYDRYIYGYDNNYVNFYEEKNEKEKEYEEEDDKASSLCENKIILSQ INCESFENDFKYYLSDYNYAFSIIDNTTNVLVAFGLYR" enhancer 1005..1308 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 1310..1509 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 1463..1483 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" primer_bind 1715..1737 /label=pCAGGS-5 /note="Chimeric intron in CAG promoter, forward primer" primer_bind 1799..1818 /label=pCAG-F /note="Rabbit beta-globin intron, for pCAG plasmids, forward primer" primer_bind 1860..1879 /label=T7 /note="T7 promoter, forward primer" promoter 1860..1878 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" protein_bind 1879..1903 /label=lac operator /bound_moiety="lac repressor encoded by lacI" /note="The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-beta-D-thiogalactopyranoside (IPTG)." promoter 1919..2028 /label=p10 promoter /note="baculovirus promoter for expression in insect cells" CDS 2052..2756 /codon_start=1 /product="monomeric derivative of DsRed fluorescent protein (Shaner et al., 2004)" /label=mCherry /note="mammalian codon-optimized" /translation="VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGT QTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFE DGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKG EIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAE GRHSTGGMDELYK" primer_bind complement(2194..2212) /label=mCherry-R /note="mCherry, reverse primer" CDS 2772..2948 /codon_start=1 /product="""Zdark"" protein, derived from the Z subunit of protein A, that binds to the dark state of LOV2 and is released upon irradiation (Wang et al., 2016)" /label=Zdk1 /translation="MVDNKFNKEKTRAGAEIHSLPNLNVEQKFAFIVSLFDDPSQSANL LAEAKKLNDAQAPK" CDS 2979..3560 /codon_start=1 /product="constitutively active mutant of human Rho family small GTPase RhoA" /label=RhoA (Q63L) /translation="MAAIRKKLVIVGDGACGKTCLLIVFSKDQFPEVYVPTVFENYVAD IEVDGKQVELALWDTAGLEDYDRLRPLSYPDTDVILMCFSIDSPDSLENIPEKWTPEVK HFCPNVPIILVGNKKDLRNDEHTRRELAKMKQEPVKPEEGRDMANRIGAFGYMECSAKT KDGVREVFEMATRAALQARRGKKKSGCLVL" CDS 3597..3629 /codon_start=1 /product="HSV (herpes simplex virus) epitope tag" /label=HSV tag /translation="QPELAPEDPED" CDS 3636..3659 /codon_start=1 /product="8xHis affinity tag" /label=8xHis /translation="HHHHHHHH" primer_bind complement(3749..3768) /label=Bglob-pA-R /note="Rabbit beta-globin polyA region, reverse primer" polyA_signal 3814..3869 /label=-beta-globin poly(A) signal /note="rabbit beta-globin polyadenylation signal (Gil and Proudfoot, 1987)" primer_bind complement(3868..3887) /label=rbglobpA-R /note="Rabbit beta-globin polyA, reverse primer. Also called rb-glob-pA-term-R" terminator 3957..4004 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" misc_recomb 4017..4722 /label=baculovirus recombination region (ORF1629) /note="contains part of ORF1629" 3'UTR 4105..4477 /label=polyhedrin 3' UTR /note="3' UTR from the Autographa californica nucleopolyhedrovirus polyhedrin gene (Suzuki et al., 2006)" CDS complement(4134..4722) /codon_start=2 /product="baculovirus capsid-associated protein" /label=ORF1629 /note="required for viral replication" /translation="IPKSSTTNLIADVLADTINRRRVAMAKSSSEATSNDEGWDDDDNR PNKANTPDVKYVQALFNVFTSSQLYTNDSDERNTKAHNILNDVEPLLQNKTQTNIDKAR LLLQDLASFVALSENPLDSPAIGSEKQPLFETNRNLFYKSIEDLIFKFRYKDAENHLIF ALTYHPKDYKFNELLKYVQQLSVNQQRTESSA" protein_bind 4731..4764 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." rep_origin complement(4932..5519) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(5011..5030) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(5532..6392) /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" primer_bind 6155..6174 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" ORIGIN 1 aactgttaca ttgcaaacgt ggtttcgtgt gccaagtgtg aaaaccgatg tttaatcaag 61 gctctgacgc atttctacaa ccacgactcc aagtgtgtgg gtgaagtcat gcatctttta 121 atcaaatccc aagatgtgta taaaccacca aactgccaaa aaatgaaaac tgtcgacaag 181 ctctgtccgt ttgctggcaa ctgcaagggt ctcaatccta tttgtaatta ttgaataata 241 aaacaattat aaatgtcaaa tttgtttttt attaacgata caaaccaaac gcaacaagaa 301 catttgtagt attatctata attgaaaacg cgtagttata atcgctgagg taatatttaa 361 aatcattttc aaatgattca cagttaattt gcgacaatat aattttattt tcacataaac 421 tagacgcctt gtcgtcttct tcttcgtatt ccttctcttt ttcatttttc tcttcataaa 481 aattaacata gttattatcg tatccatata tgtatctatc gtatagagta aattttttgt 541 tgtcataaat atatatgtct tttttaatgg ggtgtatagt accgctgcgc atagtttttc 601 tgtaatttac aacagtgcta ttttctggta gttcttcgga gtgtgttgct ttaattatta 661 aatttatata atcaatgaat ttgggatcgt cggttttgta caatatgttg ccggcatagt 721 acgcagcttc ttctagttca attacaccat tttttagcag caccggatta acataacttt 781 ccaaaatgtt gtacgaaccg ttaaacaaaa acagttcacc tcccttttct atactattgt 841 ctgcgagcag ttgtttgttg ttaaaaataa cagccattgt aatgagacgc acaaactaat 901 atcacaaact ggaaatgtct atcaatatat agttgctgat ggccggccta ttaatagtaa 961 tcaattacgg ggtcattagt tcatagccca tatatggagt tccgcgttac ataacttacg 1021 gtaaatggcc cgcctggctg accgcccaac gacccccgcc cattgacgtc aataatgacg 1081 tatgttccca tagtaacgcc aatagggact ttccattgac gtcaatgggt ggagtattta 1141 cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtcc gccccctatt 1201 gacgtcaatg acggtaaatg gcccgcctgg cattatgccc agtacatgac cttacgggac 1261 tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatgct gatgcggttt 1321 tggcagtaca ccaatgggcg tggatagcgg tttgactcac ggggatttcc aagtctccac 1381 cccattgacg tcaatgggag tttgttttgg caccaaaatc aacgggactt tccaaaatgt 1441 cgtaataacc ccgccccgtt gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat 1501 ataagcagac gtcgtttagt gaaccgtcag atcactagat gctttattgc ggtagtttat 1561 cacagttaaa ttgctaacgc cagtctcgaa cttaacgtgc agaagttggt cgtgaggcac 1621 tgggcaggta agtatcgggc cctttgtgcg gggggagcgg ctcggggctg tccgcggggg 1681 gacggctgcc ttcggggggg acggggcagg gcggggttcg gcttctggcg tgtgaccggc 1741 ggctctagag cctctgctaa ccatgttcat gccttcttct ttttcctaca gctcctgggc 1801 aacgtgctgg ttattgtgct gtctcatcat tttggcaaag aattggatcg gaccgaaatt 1861 aatacgactc actatagggg aattgtgagc ggataacaat tccccggagt taatccggga 1921 cctttaattc aacccaacac aatatattat agttaaataa gaattattat caaatcattt 1981 gtatattaat taaaatacta tactgtaaat tacattttat ttacaatcaa aggagatata 2041 ccatgtctag agtgagcaag ggcgaggagg ataacatggc catcatcaag gagttcatgc 2101 gcttcaaggt gcacatggag ggctccgtga acggccacga gttcgagatc gagggcgagg 2161 gcgagggccg cccctacgag ggcacccaga ccgccaagct gaaggtgacc aagggtggcc 2221 ccctgccctt cgcctgggac atcctgtccc ctcagttcat gtacggctcc aaggcctacg 2281 tgaagcaccc cgccgacatc cccgactact tgaagctgtc cttccccgag ggcttcaagt 2341 gggagcgcgt gatgaacttc gaggacggcg gcgtggtgac cgtgacccag gactcctccc 2401 tgcaggacgg cgagttcatc tacaaggtga agctgcgcgg caccaacttc ccctccgacg 2461 gccctgtaat gcagaagaag actatgggct gggaggcctc ctccgagcgg atgtaccccg 2521 aggacggcgc cctgaagggc gagatcaagc agaggctgaa gctgaaggac ggcggccact 2581 acgacgctga ggtcaagacc acctacaagg ccaagaagcc cgtgcagctg cccggcgcct 2641 acaacgtcaa catcaagttg gacatcacct cccacaacga ggactacacc atcgtggaac 2701 agtacgaacg cgccgagggc cgccactcca ccggcggcat ggacgagctg tacaagggtt 2761 ctggaagttc catggtggat aacaaattca ataaagaaaa gacgcgtgcc ggtgcggaaa 2821 tccattctct gccaaacctg aatgttgagc agaagtttgc cttcatcgtg agcctgtttg 2881 atgatccatc tcagagcgcc aatctgctgg ccgaagccaa aaaactgaac gatgcccagg 2941 ccccaaaagg atccggtggt tctggttctg gtggtaccat ggctgccatc cggaagaaac 3001 tggtgattgt tggtgatgga gcctgtggaa agacatgctt gctcatagtc ttcagcaagg 3061 accagttccc agaggtgtat gtgcccacag tgtttgagaa ctatgtggca gatatcgagg 3121 tggatggaaa gcaggtagag ttggctttgt gggacacagc tgggctggaa gattatgatc 3181 gcctgaggcc cctctcctac ccagataccg atgttatact gatgtgtttt tccatcgaca 3241 gccctgatag tttagaaaac atcccagaaa agtggacccc agaagtcaag catttctgtc 3301 ccaacgtgcc catcatcctg gttgggaata agaaggatct tcggaatgat gagcacacaa 3361 ggcgggagct agccaagatg aagcaggagc cggtgaaacc tgaagaaggc agagatatgg 3421 caaacaggat tggcgctttt gggtacatgg agtgttcagc aaagaccaaa gatggagtga 3481 gagaggtttt tgaaatggct acgagagctg ctctgcaagc tagacgtggg aagaaaaaat 3541 ctgggtgcct tgtcttgtaa aagcttgcgg ccgcacagct gtatacacgt gcaagccagc 3601 cagaactcgc cccggaagac cccgaggatc tcgagcacca ccatcaccat caccatcact 3661 aagtgattaa cctcaggtgc aggctgccta tcagaaggtg gtggctggtg tggccaatgc 3721 cctggctcac aaataccact gagatcgatc tttttccctc tgccaaaaat tatggggaca 3781 tcatgaagcc ccttgagcat ctgacttctg gctaataaag gaaatttatt ttcattgcaa 3841 tagtgtgttg gaattttttg tgtctctcac tcggaaggac atatgggagg gcaaatcatt 3901 taaaacatca gaatgagtat ttggtttaga gtttggcaac atatgcccat atgtaactag 3961 cataacccct tggggcctct aaacgggtct tgaggggttt tttgctgaaa gcatgcggag 4021 gaaattctcc ttgaagtttc cctggtgttc aaagtaaagg agtttgcacc agacgcacct 4081 ctgttcactg gtccggcgta ttaaaacacg atacattgtt attagtacat ttattaagcg 4141 ctagattctg tgcgttgttg atttacagac aattgttgta cgtattttaa taattcatta 4201 aatttataat ctttagggtg gtatgttaga gcgaaaatca aatgattttc agcgtcttta 4261 tatctgaatt taaatattaa atcctcaata gatttgtaaa ataggtttcg attagtttca 4321 aacaagggtt gtttttccga accgatggct ggactatcta atggattttc gctcaacgcc 4381 acaaaacttg ccaaatcttg tagcagcaat ctagctttgt cgatattcgt ttgtgttttg 4441 ttttgtaata aaggttcgac gtcgttcaaa atattatgcg cttttgtatt tctttcatca 4501 ctgtcgttag tgtacaattg actcgacgta aacacgttaa atagagcttg gacatattta 4561 acatcgggcg tgttagcttt attaggccga ttatcgtcgt cgtcccaacc ctcgtcgtta 4621 gaagttgctt ccgaagacga ttttgccata gccacacgac gcctattaat tgtgtcggct 4681 aacacgtccg cgatcaaatt tgtagttgag ctttttggaa ttgcgatcgc ataacttcgt 4741 atagcataca ttatacgaag ttataagctc ggaacgctgc gctcggtcgt tcggctgcgg 4801 cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac 4861 gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg 4921 ttgctggcgt ttttccatag gctccgcccc ctgacgagca tcacaaaaat cgacgctcaa 4981 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct 5041 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc 5101 cttcgggaag cgtggcgctt tctcaatgct cacgctgtag gtatctcagt tcggtgtagg 5161 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct 5221 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag 5281 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga 5341 agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga 5401 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg 5461 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag 5521 aagatccttt gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt 5581 cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta 5641 ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta 5701 tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc 5761 gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat 5821 agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt 5881 atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg 5941 tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca 6001 gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta 6061 agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg 6121 cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact 6181 ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg 6241 ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt 6301 actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga 6361 ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc 6421 atttatcagg gttattgtct catgtccgcg cgtt //